The present invention relates to novel purified hydroxylase components of methane monooxygenase enzymes found in methanotrophic bacteria, a novel process for purifying the hydroxylases and a novel process for oxidizing hydrocarbons using purified hydroxylase, molecular oxygen (O.sub.2) and commercially available reducing agents.
Alkanes are among the most unreactive carbon compounds. Alkane reactions have characteristically high activation energies and often form products which are more reactive than the parent hydrocarbons. The reactions are difficult to control for selective production of intermediate and end products in high yield. The oxidation of methane is an example of the difficulties inherent in alkane reactions.
Methane reactions are of particular interest because large quantities of natural gas are located in remote areas, far from their main centers of consumption. It is expensive, however, to transport the gas to most locations where it is used. Current technology for methane conversion is based on steam reforming, followed by either the Fischer-Tropsch process or by methanol synthesis. It is hoped that more direct paths to either product--methanol or higher hydrocarbons--can be found which are more efficient, more selective and less costly. Other alkanes are also abundant and relatively inexpensive. New technologies would enable these and other hydrocarbons to be used as inexpensive feedstocks for the synthesis of commodity chemicals as well as precursors to liquid fuels.
Additionally, the increasing presence of hazardous substances such as benzene, p-xylene and substituted hydrocarbons such as trichloroethylene in waste water streams demonstrates a need for a means for converting these substances into other substances which are less hazardous or not hazardous at all. As a result, researchers have looked to a variety of means for converting hydrocarbons into other useful products.
In particular, many researchers have attempted to mimic naturally-occurring biological systems. Certain bacteria have been found which rely exclusively on methane as their source of life-sustaining carbon compounds and energy. The first and most difficult step in the processing of methane by these methanotrophic bacteria is its conversion into methyl alcohol. This conversion of methane to methanol is catalyzed by a family of enzymes now known as methane monooxygenases.
Methane monooxygenases utilize molecular oxygen as their oxygen source. Moreover, although methane is the only hydrocarbon known to sustain growth of the bacteria, methane monooxygenases are able to catalyze the oxidation of numerous saturated and unsaturated hydrocarbons. Oxidation is accomplished by forming an activated oxygen:enzyme:substrate complex charged with two electrons from a suitable donor, such as NADH. The iron in methane monooxygenases is present in the form of a .mu.-oxo bridged binuclear iron center. Thus, the monooxygenases have two irons available for reaction. Other than the methane monooxygenases, no other proteins containing oxo bridged iron are known to catalyze oxygenase reactions.
Methanotrophic bacteria which utilize methane monooxygenases are classified as Type I or Type II based on morphological differences in their membrane-fine structure and divergence in their metabolic pathways. Both Type I and II methanotrophs are able to express methane monooxygenases that are either soluble or membrane bound. The monooxygenase that is expressed is determined by the conditions employed for bacterial growth. Little is known about the membrane-bound monooxygenases, but some of the soluble enzymes have been isolated and examined. Examples include the methane monooxygenases isolated from the Type I organism, Methylococcus capsulatus (Bath); the Type II organisms, Methylobacterium organophilum and Methylobacterium sp. (CRL-26); and the Type II organism, Methylosinus trichosporium OB3b, the study of which led to the present invention. Those examined to date appear to be similar in composition and function.
Most methane monooxygenases isolated to date are comprised of three proteins which are nominally designated in the literature as components A, B and C. Components A and C are also referred to as hydroxylase and reductase components, respectively, because of the roles they are perceived to play in bacterial oxidation. While others have isolated the components from various strains of methanotrophic bacteria, those isolated prior to the present invention are characterized by low specific activities. Woodland et al., for example, have reported a final specific activity of 72 nmol/min/mg for the hydroxylase from the Type I organism, Methylococcus capsulatus (Bath) (J. Biol. Chem. 259, 53-59, 1984); and Patel et al. have reported a final specific activity of 208 for the hydroxylase from the Type II organism, Methylobacterium sp. (CRL-26) (J. Bact. 169, 2313-2317, 1987). These specific activities are much less than the approximately 800 nmol/min/mg specific activity observed for in vivo oxidation of methane.
Methane monooxygenases appear to catalyze hydrocarbon oxidation in an orderly manner. Normally, the hydrocarbon to be oxidized, oxygen and donated electrons collect at various sites of the monooxygenase system before oxidation occurs. More particularly, the reductase is believed to accept donated electrons. Component B is believed to mediate the transfer of electrons from the reductase to the hydroxylase where the oxidation is believed to occur. Previous studies by Dalton and Woodland have suggested that the substrate binds to the hydroxylase (Adv. Appl. Micro. 26, 71-87, 1980; J. Biol. Chem. 259, 53-59, 1984). The suggestion remained unconfirmed, however.
All three components are reportedly required for bacterial oxidation for Methylococcus capsulatus (Bath) (Colby et al., Biochem. J. 177, 903-908, 1979) and Methylosinus trichosporium OB3b (Fos et al., Biochem. Biophys. Res. Comm. 154, 165-170, 1988) Component B is reportedly not required for bacterial oxidation by Methylobacterium sp. (CRL-26) (Patel et al., J. Bact. 169, 2313-2317, 1987; Patel, Arch. Biochem. Biophys. 252, 229-236, 1987) and Methylobacterium organophilum (Patel et al. U.S. Pat. No. 4,587,216). Patel has disclosed the use of purified hydroxylase and reductase proteins from various organisms in combination with a cofactor system comprising NADH or NADPH for oxidizing hydrocarbons (U.S. Pat. No. 4,587,216).
Some researchers have attempted to mimic oxidation by methane monooxygenases using model compounds containing binuclear iron groups. Vincent et al., for example, have disclosed the oxidation of hydrocarbons using the model complex Fe.sub.2 O(OAc).sub.2 Cl.sub.2 (bipy).sub.2 where bipy is 2,2'-bipyridine (J. Am. Chem. Soc. 110, 6898-6900, 1988). Oxidation was accomplished using Bu.sup.t OOH and, alternatively, O.sub.2 as the monooxygen transfer reagent. The latter system also employed Zn powder and glacial acetic acid as electron and proton donors. Kitajima et al. have used synthetic analogues of hemerythrin to oxidize hydrocarbons (J. Chem. Soc. Chem. Comm. 7,485-486, 1988). O.sub.2 was used as the oxygen source in the presence of Zn powder and glacial acetic acid. Murch et al. have used (Me.sub.4 N) [Fe.sub.2 L(OAc).sub.2 ].sup.5 where L is N,N'-(2-hydroxy-5-methyl-1,3-xylene)bis(N-carboxymethylglycine) (J. Amer. Chem. Soc., 108, 5027-5028, 1986). H.sub.2 O.sub.2 was used as the oxygen source. However, the oxidation rates obtained using these systems are low and none have been shown capable of large scale oxidation of hydrocarbons.
Although native methane monooxygenase systems are able to oxidize hydrocarbons, they do not present viable commercial alternatives to the more conventional methods for oxidation. Native systems obtain the electrons needed for oxygen activation from such biochemicals as NADH and NADPH which are labile and expensive and, therefore, unsuitable for production at a commercial scale. Since most native systems comprise three protein components which must be bound together, they are also too complex for efficient operation at commercial scale. Similarly, the two-component system disclosed be Patel also uses expensive and labile biochemicals. Moreover, even the use of two components presents difficulties for oxidations at commercial scale.
Accordingly, it is an object of the present invention to provide a novel method for extracting novel purified hydroxylase components with high specific activity from soluble methane monooxygenases.
It is another object of the present invention to provide a novel method for oxidizing hydrocarbons. More particularly, it is an object of this invention to provide a novel method for oxidizing hydrocarbons using molecular oxygen, chemical reductants or electrochemical or photochemical means for supplying electrons in the presence of an electron transfer-mediating compound, such as methyl viologen, and the purified hydroxylases obtained from the soluble methane monooxygenase.
It is still another object of this invention to provide a novel method for oxidizing hydrocarbons using purified, high activity hydroxylase from the methanotroph Methylosinus trichosporium OB3b, proflavin, methyl viologen and sodium dithionite in the absence of reductase and component B proteins.
Other objects, advantages and novel features of the invention will be apparent from the Description below.